The
prevailing paradigm ofinfectious disease is based on the work of Koch and
colleagues, who more than 150 years ago isolated individual strains of bacteria
and developed the pure culture-method that is still used today. That work
enlightened medicine by firmly establishing the germ-theory of transmissible
diseases and demonstrated that diseases like dysentery, tuberculosis and anthrax
are caused by microbiological agents.1 Hence, the field of microbiology
developed around Koch¢s methods with clinical microbiologists working
overwhelmingly with pure log-phase cultures in nutrient-rich media because this
approach provided such a powerful tool for the study of acute epidemic bacterial
diseases. However, this approach that examines only planktonic bacteria
(free-floating, single cell phenotype) may have limited development of a more
thorough understanding of microbial processes. In most natural environments and
in chronic bacterial infections, the planktonic phenotype generally exists only
transiently and usually as a minor population. Emerging evidence describes
bacterial populations as predominantly poly-microbial, sessile, community-based
aggregations embedded in a self-secreted matrix that provides numerous
advantages for persistence in the face of environmental and
host-challenges.Therefore, biofilms and the existence of a complex bacterial
life-cycle provide a new perspective through which to view infectious diseases.
Much of the support for this perspective has come about through the application
of new detection- andvisualization-methods that have provided evidence for the
theory that chronic infections are fundamentally different than acute infections
and that different interventional approaches are necessary to treat these
biofilm-infections more efficiently.

What
Is a Biofilm?

A
biofilm is a thin layer of micro-organisms that adhere to the surface of an
organic or inorganic structure, together with their secreted polymers. Biofilms
are the predominant phenotype of nearly all bacteria in their natural habitat,
whether pathogenic or environmental. The biofilm provides a bulwark against
environmental stressors and can include organisms from multiple kingdoms as in
the case of mixed bacterial-fungal biofilms. Thirty years ago, Costerton et al.2
was the first to examine the attributes of biofilms, examining the extracellular
polymeric substances (EPS) that holds these community bacteria together. He and
other researchers have since implicated biofilms in diverse a-biotic and biotic
systems, including oil-pipelines, hot-tubs, teeth and mucosa. Thus biofilms,
with their community-defenses, are a ubiquitous feature of bacteria in nature
and in some bacterial infections. The resident bacterial community in a biofilm
has added defenses and multiple mechanisms for survival, such as defenses
against phagocytosis, UV-radiation, viral attack, shear stress and dehydration,
as well as against biocides, antibiotics and host-immunity. Biofilms have
demonstrated the ability to persist in 100 to 1000 times the concentrations of
antibiotics and biocides that can inhibit planktonic cells.3 Similarly,
granulocytes, macrophages and other phagocytes are unable to engulf a biofilm as
they would individual planktonic cells. The genotypic and phenotypic diversity
of the biofilm allow adaptation to overcome multiple stresses and to survive
most sequential therapies. The hallmark of biofilms is genetic and phenotypic
diversity, which enhances the robustness of the bacterial community.4 An
increasing body of evidence suggests that laboratory-cultivated bacteria share
few characteristics with infectious biofilms. When bacteria naturally transition
from individual planktonic cells to a community of biofilm-tissue, the
transcriptional expression of the bacteria radically changes. Although this
phenotypic transition is occurring, the bacteria are excreting protective
polymers and incorporating environmentalmolecules that bind the bacterial
community to a surface and to each other. The biofilm bacterial community uses
secreted pheromones (e.g. quorum-sensing molecules) and other molecules for
cell-cell signaling, even between species. These coordinated activities render
the biofilm a formidable opponent for the host.

Biofilms
as a Novel Focus.

There
are 3 significant limitations of continuing to operate within the planktonic
paradigm.

-
First, because planktonic cells have fewer defenses than biofilm-communities, a
treatment such as an antibiotic might provide excellent in vitro test-results,
when tested on planktonic cells only, but poor in vivo results, in which the
biofilm-phenotype may predominate. The same strain of bacteria can be hundreds
or thousands of times more resistant to antibiotics if part of a biofilm
community.3,5 This planktonic bias undoubtedly accounts for at least some of the
discrepancy that can occur between in vitro test results and in vivo response to
antibiotic therapy.6

-
Second, current culture-methods do not identify the majority of bacteria now
known to be in host-infections.7 Researchers have developed molecular, genomic,
metagenomic, transcriptomic and proteomic methods because they determined that
only approximately 1% of the cells they observed in ecosystems actually produced
colonies by conventional culture-methods.8 The conclusion of these DNA-based and
RNA-based studies is that culture-methods detect only a small minority of
organisms actually present in natural and pathogenic bacterial communities.
Therefore, treatment based on conventional cultures may address only 1 or 2
bacterial species in a complex biofilm-community that may include dozens of
other species of bacteria, or even of fungi.

-
Third, planktonic techniques such as culturing may lead to an inaccurate or
incomplete diagnosis because cultures do not detect biofilm-cells that are
viable but not culturable. Diseases that yield only intermittent positive
cultures (e.g. otitis media, prostatitis) have been suggested to be ¡sterile
inflammations¢ or viral infections.9 An example of a serious result from
reliance on standard cultures is that revisions of the Sulzer acetabular cup
continue to be treated as ¡sterile loosenings¢, although this process probably
represents a biofilm-infection that does not yield positive cultures but can be
detected by molecular methods.10

New
Perspectives, Options for Detection and Treatments

Changing
the perspective about chronic infectious disease to include biofilm enables 2
important insights. First, it opens new methods for detection and treatment.
Second, it provides a global reconceptualization of many chronic infectious
diseases as resulting from a biofilm, allowing biofilm-principles to be shared
across disciplines. Recent studies have investigated new methods for detecting
the components of a biofilm. Several investigations have used modern molecular
methods, such as denaturing gradient gel electrophoresis and denaturing
high-performance liquid chromatography, along with imaging techniques including
fluorescent in situ hybridization. Also, molecular methods such as polymerase
chain reaction (PCR) and pyro-sequencing in conjunction with conventional
culture-methods have been used to determine the bacterial species composition of
chronic infections.7 Performing molecular tests as part of routine bacterial
analysis is becoming a real option for clinical laboratories. These tests could
include methods such as PCR, reverse transcriptase PCR, micro-arrays,
antigen-testing and rapid sequencing. Only a few of these methods are being used
to test for certain pathogens but culture-free identification of all pathogens
and their corresponding resistance-markers may soon become routine.11 A biofilm
focus also provides new strategies for treatment of chronic infections.
Biofilm-based treatments might
block initial bacterial attachment to a surface, block or destroy EPS-formation,
interfere with cell-cell signaling pathways and use bacteriostatic or
bactericidal agents at the same time. Concomitant therapies that not only
attempt to eradicate bacteria but also affect the biofilm¢s community-structure
and communications may prove more effective than a single or sequential strategy
such as antibiotic therapy.4 This multimodality approach to therapy is commonly
used in other areas of medicine, such as the treatment of human immunodeficiency
virus for which combination antiretroviral therapy is used to achieve the best
clinical outcome.

Chronic
Bacterial Disease as Biofilm Disease

Another
main benefit the biofilm-model allows is the re-conceptualization of multiple
chronic diseases as biofilm-diseases. Biofilm-disease has been viewed as various
diseases that affect a variety of tissues and structures, including ear, nose,
throat, mouth, eye, lung, heart, kidney, gall-bladder, pancreas, nervous system,
skin, bone, as well as virtually every implanted medical device. The Centres for
Disease Control and Prevention estimates that more than 65% of infections are
caused by bacteria growing in biofilms12 and Lewis13 suggests that the
proportion is 80% - by far the majority of infections are estimated to be caused
by biofilms. Thus, when biofilm-infections are combined into a single disease
category, the prevalence of the disease is significant and the mortality
associated with biofilm disease is substantial. Additionally, the
disease-processes and management-strategies of biofilms are related. For
example, the biofilm-diseases of cystic fibrosis, pneumonia and an infected knee
replacement prosthesis are different, but strategies used to manage the biofilm
in the lung will be similar to the strategies used for the biofilm on an
infected knee-implant.

Typical
and Familiar Biofilms

The
typical biofilm-disease manifests with common features. The initial infection is
subtle and usually not immediately life-threatening, and antibiotics usually are
prescribed. Subsequent exacerbations occur and are again usually treated with
antibiotics and adjunct therapies such as corticosteroids. However, the
infection worsens when treatment is withdrawn. If the patient¢s clinical status
worsens or if the disease progresses to the point that function of the affected
tissue or device is sufficiently impaired, a decision often is made to remove
the infected tissue or component by surgery. The goal of the physician when
addressing the disease is to manage the symptoms and signs, and to suppress
exacerbations with the understanding that disease eradication is unlikely
without surgical intervention. Perhaps the most commonly encountered
biofilm-disease is dental plaque, a condition that can be managed effectively by
dental professionals. Periodontal disease and tooth-loss have greatly diminished
over the last century due to the multiple concurrent strategies espoused by
dentists. The suppression of biofilm in the oral cavity begins with frequent
removal of the biofilm by daily brushing and flossing, coupled with periodic
dental visits for plaque removal. If the bacterial biofilm load is too great and
disease progresses, the frequency of biofilm-management is increased in an
attempt to overcome the disease. Dentistry has successfully confronted
biofilm-disease and similar approaches may be used as a model for medicine to
manage chronic infections.

Conclusions

Treating
chronic infectious diseases cannot be limited to infectious disease specialists
any more than the management of diabetes can be limited to endocrinologists. By
shifting away from the planktonic model of microbiology to the biofilm-model,
new methods for detection and treatment become available. Because of molecular
methods, science now has the ability to detect biofilms and understand the
implications of interspecies chaos that contribute to infections. With these new
scientific approaches along with coordination of clinical and laboratory
efforts, education and research, it is possible to imagine overcoming much of
biofilm-disease.

Background:
In a March, 2005 press article, Nanobac Pharmaceuticals, Inc. stated that its
urology researchers have shown a causal relationship between Nanobacteria
infection and urological disorders such as chronic prostatitis, kidney stones,
and polycystic kidney disease (PKD). Until these very studies, no infection,
either viral or bacterial had ever been shown to be indicated as a cause of
these diseases. Because of the debilitating nature of these urological diseases,
and Nanobac researchers' knowledge that Nanobacteria played a key role in their
development, Nanobac has focused and continues to focus major efforts in further
defining the relationship between Nanobacteria infection and these diseases, the
development of diagnostic tests for nanobacterial infections in these diseases
and the development of therapies to effectively treat these diseases.

Chronic
Prostatitis:
A recent observational study of chronic prostatitis (CP) patients, conducted by
urology's top prostatitis researcher Daniel Shoskes, M.D (Cleveland Clinic),
demonstrated significant improvement in the symptoms of chronic
prostatitis/chronic pelvic pain syndrome for those patients who used 3 months of
Nanobac's patented nanobiotic treatment. The treated group of fifteen patients
had suffered from prostate stones and longstanding Chronic Pelvic Pain Syndrome
(CPPS) that had been unresponsive to all prior known medical therapies.
Nanobac's nanobiotic treatment is designed to eliminate the stone-forming
Nanobacteria. Shoskes' study found that 80% of the patients with Chronic
Prostatitis had significant improvement in symptoms after only 3 months
treatment. Two patients who had been on complete medical disability have
returned to work. The Nanobac nanobiotic treatment regimen is currently
available.

Kidney
stones & Astronauts:
Kidney stones can be an excruciatingly painful and debilitating, are known to
affect over 20 million Americans and do recur in 50% of patients within 5 years.
Several studies conducted by prominent medical researchers worldwide have shown
Nanobacteria as the causative culprit in kidney stone formation. Depending on
the patient population, researchers have reported that up to 97% of kidney
stones have Nanobacteria. Since Nanobacteria create calcium deposits (stones) in
tissue and are physically present in the kidney stones, they are therefore
thought to be the initiating core (nidus) of kidney stones. This year, research
by Neva Ciftcioglu, PhD (Director of Nanobacteria Research at NASA-Houston) and
her NASA scientists published a study showing Nanobacteria infections to grow
faster in space flight simulated conditions (zero gravity) than here on Earth.
This finding explains why Astronauts frequently develop kidney stones and why
they are particularly at risk for kidney stones on long duration missions.
Ciftcioglu's NASA team is working with Nanobac on this application of
nanobiotics and nanobacterial diagnostics as well as other applications.

Polycystic
Kidney Disease (PKD):
Polycystic kidney disease (PKD) is a genetic disorder characterized by the
growth of numerous cysts in the kidneys. The cysts are filled with fluid (think
small water balloons). The PKD cysts can slowly replace much of the mass of the
kidneys, the pressure eventually reduces kidney function and ultimately leads to
kidney failure. PKD is the number one cause for the need of kidney transplants.
Research has shown that in PKD patients up to 100% of kidney cyst fluids and
urine were positive for Nanobacteria. Because Nanobacteria are present in PKD
patients, Nanobac is prepared to provide diagnostic tests for Nanobacteria
infections and their Nanobiotics to treat the infections.

About
Nanobac Pharmaceuticals:
Nanobac Pharmaceuticals' mission is to improve people's health through the
detection and eradication of Nanobacteria infections. Their pioneering research
is firmly establishing the pathogenic roles of Nanobacteria in a broad arena of
tissue calcifications, particularly as found in coronary artery disease, heart
disease, heart valve disease, vascular disease, kidney diseases and cancers.
Nanobac has isolated two biomarkers of nanobacterial infection and has developed
multiple nanobacteria tests. Nanobac has developed diagnostic tests for the
detection of Nanobacteria in blood, serum, urine and spinal fluids and has
received European (EU) approval of its NB2=99 ELISA assays that detect
Nanobacterial infections using Antigen and IgG Antibodies to Nanobacteria.
Nanobac is also seeking FDA approval of these diagnostic tests for use in the
United States. Nanobac exclusively holds a series of US and foreign Patents that
cover the diagnostic testing of and treatment of nanobacterial infections.
Nanobac is currently developing the first FDA approved Nanobiotics for the
treatment of Nanobacterial infections in different disease states. Nanobac
currently markets a patented Nanobiotic treatment and its nanobacterial tests
are currently available to physicians in research.

Nanobac
Introduces New Improved ELISA Kits for Detection of Calcifying Nano-Particles,
also known as Nanobacteria

0/26/2005
8:00:00 AM EST Nanobac Life Sciences, Inc. (OTCBB: NNBP) ("Nanobac" or
"the Company") announced today the introduction of two new versions of
its NANOCAPTURE(TM) and NANO-SERO(TM) ELISA test kits for the detection of
Calcifying Nano-Particles (CNPs), also known as nanobacteria, in serum or
plasma. The new kits offer improved sensitivity, lower background and improved
stability over the previous versions. Nanobac has added new standards to improve
precision at the low end and freeze dried the critical components for improved
stability. "Our demand for CNP diagnostic products is increasing as CNP's
or nanobacteria are implicated in more and more chronic
diseases," stated
Grant Carlson, Nanobac's President and Chief Operating Officer. "Nanobac
has worked hard to develop robust, sensitive and reproducible ELISA test kits
for the CNP/nanobacteria research community.

These
kits are CE marked and ready for immediate shipment throughout the European Union.
In the United States the kits will be sold For Research Use Only (RUO)."
The NANOCAPTURE ELISA is a two-site immunoenzymatic assay for the quantitative
measurement of CNP/nanobacterial antigen in serum or plasma. The NANO-SERO ELISA
is also a two site immunoenzymatic assay for the quantitative measurement of
anti-CNP antibodies in serum or plasma.

Nanobac
will feature the NANOCAPTURE and NANO-SERO ELISA test kits at the world's #1
medical trade show, MEDICA, in Dusseldorf, Germany, November 16-19, 2005. Last
year the show attracted 136,000 visitors from around the world. Nanobac intends
to meet with potential customers and establish its global network of in vitro
diagnostic distributors.

The
laboratory diagnosis of acute bacterial prostatitis is straightforward and
easily accomplished in clinical laboratories. Chronic bacterial prostatitis, and
especially chronic idiopathic prostatitis (most often referred to as abacterial
prostatitis), presents a real challenge to the clinician and clinical
microbiologist.

Clinically,
the diagnosis of chronic idiopathic prostatitis is differentiated from that of
acute prostatitis by a lack of prostatic inflammation and no
"significant" (controversial) leukocytes or bacteria in the expressed
prostatic secretions. Despite these diagnostic criteria, the etiology of chronic
idiopathic prostatitis is unknown. While this review covers the entire spectrum
of microbially caused acute prostatitis (including common and uncommon bacteria,
viruses, fungi, and parasites) and microbially associated chronic prostatitis, a
special focus has been given to chronic idiopathic prostatitis. The idiopathic
syndrome is commonly diagnosed in men but is poorly treated. Recent data
convincingly suggests a possible bacterial etiology for the condition.

Provocative
molecular studies have been published reporting the presenceof 16S rRNA
bacterial sequences in prostate biopsy tissue that is negative for ordinary
bacteria by routine culture in men with chronic idiopathic prostatitis.
Additionally, special culture methods have indicated that difficult-to-culture
coryneforms and coagulase-negative staphylococci are present in expressed
prostatic secretions found to be negative by routine culture techniques.

Treatment
failures are not uncommon in chronic prostatitis. Literature reports suggest
that antimicrobial treatment failures in chronic idiopathic prostatitis caused
by organisms producing extracellular slime might result from the virulent
properties of coagulase-negative staphylococci or other bacteria. While
it is difficult to definitively extrapolate from animal models, antibiotic
pharmokinetic studies with a murine model have suggested that treatment failures
in chronic prostatitis are probably a result of the local microenvironment
surrounding the persistent focal and well-protected small bacterial biofilms
buried within the prostate gland. These conclusions support the molecular and
culture data implicating bacteria as a cause of chronic idiopathic prostatitis.

PURPOSE:
Category III chronic prostatitis/chronic pelvic pain syndrome (CPPS) is a common
debilitating condition of unclear etiology. Patients often have prostatic
calcifications but a link to symptoms is controversial. Nanobacteria are
implicated in stone formation in the urinary tract and, therefore, therapy to
eliminate nanobacteria and the stones that they produce might have an impact on
CPPS symptoms.

MATERIALS
AND METHODS: A total of 16 men with recalcitrant CPPS refractory to multiple
prior therapies were treated with comET (Nanobac Life Sciences, Tampa, Florida),
which consists of 500 mg tetracycline, a proprietary nutraceutical and an
ethylenediaminetetraacetic acid suppository daily. The National Institute of
Health Chronic Prostatitis Symptom Index (NIH-CPSI), transrectal ultrasound, and
blood and urine tests for nanobacterial antigen were performed at the start and
conclusion of 3 months of therapy. One patient was lost to followup.

RESULTS:
Mean NIH-CPSI total score +/- SD decreased from 25.7 +/- 1.6 to 13.7 +/- 2.0
(p <0.0001). Significant improvement was seen in each subscore domain. A
total of 12 patients (80%) had at least 25% improvement on NIH-CPSI and 8 (53%)
had at least 50% improvement. Nanobacterial
antigen or antibody was found in 60% of serum and 40% of urine samples. In 10
patients who underwent transrectal ultrasound after therapy prostatic stones
were decreased in size or resolved in 50%.

CONCLUSIONS:
Therapy designed to eliminate nanobacteria resulted in significant improvement
in the symptoms of recalcitrant CPPS in the majority of men, whether due to the
treatment of stone producing nanobacteria or through some other mechanism.
Prospective placebo controlled trials are warranted.

Nanobac Pharmaceuticals, Inc.
(OTCBB:NNBP) ("Nanobac" or "the Company") today announced
that Inflammatory nanoparticles that produce calcified deposits like those found
in arthritic joints have been cultured from the synovial fluid of rheumatoid
arthritis and osteoarthritis patients by Japanese researchers, as reported in
the Journal of Proteome Research.

"This is the first report to
indicate that human synovial fluids contain Nanobacteria-like particles,"
the study notes. The study, Nanobacteria-Like Particles in Human Arthritic
Synovial Fluids, by T. Tsurumoto, T. Matsumoto, A. Yonekura, and H. Shindo,
Department of Orthopaedics, Graduate School of Biomedical Sciences, Nagasaki
University, supports the existence and pathogenic role of nanobacteria-like
particles found in human diseases such as heart, prostate
and kidney disease by Mayo Clinic and other researchers. In a two year
experiment, calcifying nanoparticles were cultured from the joint fluid of each
participating patient, demonstrating that 100 percent of patients had the
particles. The experiment was based on methods pioneered by Nanobac Scientists,
Drs. Neva Ciftcioglu and Olavi Kajander.

"After
about 2 months of culture, nanoparticles appeared in the
synovial fluids from all the patients to greater or lesser degrees," the
study found. "These nanoparticles gradually increased in number and in
size." Medical researchers have often theorized about the presence of a
calcifying agent that generates associated painful inflammation in arthritis,
but until now had never found one. Nanobacteria produce a calcium phosphate
material known as calcification, which is shown in many studies, and cited in
the Merck Manual of Diagnosis and Therapy, as provoking inflammation. "If
self-proliferating nanoparticles exist in mammalian synovial fluids and
membranes, then they may have an effect on many joint diseases," the
study's authors concluded. "This is another example of independent
researchers finding nanobacteria in patients with a specific disease
condition," said Nanobac Co-Chairman Dr. Benedict Maniscalco, Fellow of the
American College of Cardiology (FACC). "It shows the value of our research,
which is the only work to explain how calcification occurs in diseases
afflicting most of the aging population," Dr. Maniscalco added.

The findings come on the heels of a
paper published in the World Journal of Urology by Cleveland Clinic researcher
Dr. Daniel Shoskes and Dr. Hadley M. Wood, which
concluded that nanobacteria-like particles may play a ubiquitous role in
prostate disease. Another independent paper published earlier this
year in the journal Urology Research by Khullar et al reports induction of renal
calcification by nanobacteria. Disclosure statement: Nanobac Pharmaceuticals did
not fund the arthritis or Khullar studies, so the findings are
independent.

About Nanobac Pharmaceuticals
Nanobac Pharmaceuticals, Inc. is a life science company dedicated to the
discovery and development of products and services to improve people's health
through the detection and treatment of Calcifying Nanoparticles, otherwise known
as "nanobacteria". The Company's pioneering research is establishing
the pathogenic role of nanobacteria in soft tissue calcification, particularly
in coronary artery, prostate, and vascular disease. Nanobac's drug discovery and
development is focused on developing new and existing compounds that effectively
inhibit, destroy or neutralize CNPs. Nanobac manufactures In Vitro Diagnostic
(IVD) kits and reagents for the detection of Calcifying Nanoparticles. IVD
products include the NANOCAPTURE(TM) and NANO-SERO(TM) ELISA assays and the
Nano-Vision(TM) line of antibodies and reagents. Nanobac's BioAnalytical
Services works with biopharmaceutical partners to develop and apply methods for
avoiding, detecting, and inactivating or eliminating CNPs from raw materials.

Bacteria
are ubiquitous in our environment.The vast majority of bacteria do not live in a free-swimming
planktonic form, but rather in the self-produced, protective environment of a
biofilm, which seems to explain why some infections are nearly impossible to
eradicate.Spend a weekend
unclogging bathroom pipes or slipping on the rocks in a mountain stream and you
will understand some of the other ways biofilms affect us.

The
concept of biofilms is relatively new -William Costeiton coined the term in
1978.His early studies
demonstrated the protective mechanisms of biofilms in providing an environment
for bacteria away from white cells, antibacterials, and environmental stresses.His microscopic
examination of biofilms identified bacterial colonies
interwoven with a polysaccharide matrix -a previously unappreciated fact.Small channels in the biofilm matrix permit the flow of oxygen,
nutrients, and waste necessary to maintain microbial activity and reproduction.

Scanning
electron micrograph
of a Staphylococcus biofilm
on the inner surface of a
needleless connector.

While
we have effective antibiotics to kill planktonic bacteria, the reservoir of
bacteria contained in biofilms does not respond well to current antibiotics
because of the protective nature of these structures.The large clusters of bacteria that make up a biofilm are similar to
chemotherapy-resistant malignant cells, in that organisms in the clusters'
center are protected from antibacterial invasion by a "wall" of fellow
bacteria.Additional research has
pointed to biofilm as producing a slower bacterial metabolism,
antibiotic-degrading enzymes, and even the ability to "pump"
antibacterial agents out of the biofilm before they can have any effect on the
bacteria.Indeed, bacteria in a
biofilm environment can be up to 1,000 times more resistant to antibiotics than
the same bacteria circulating in a planktonic state.*

Uropathogens
and other bacteria produce biofilms unique to their species.In the lung, for example, Pveudomonas
aeruginosa use their flagella to attach to each other and to the organ
surface, thereby initiating colony formation.Staphylococcus, a major biofilm-forming organism (see the photo),
produces infections in wounds and on prosthetic devices such as penile
prostheses and artificial urinary sphincters, and may also be involved in the
initiation of catheter-borne infections.Each
year, biofilm-related infections on catheters, prosthetic devices, urinary
catheters and tubes, and contact lenses cost the medical industry billions of
dollars.The Centers For Disease
Control estimate that more than 65% of hospital-acquired infections have
biofilms as an integral part of their morbidity and potential mortality.
Prosthetic implant infections are an excellent ease in point.Even when antibiotic agents are used in high doses and are in direct
contact with the prosthetic device, these difficult, high-morbidity infections
are rarely eradicated, necessitating removal of the device and its accompanying
bacteria-filled biofilm.

The
search for new antibiotic agents is admirable.However, the key to conquering complex urologic infections may lie
instead in overcoming the bacteriaprotecting barrier effect of biofilm.Research on antibiofilm compounds is underway at a number of laboratories
across the country, and in vitro models are now available to identify the
structure, architecture, unique characteristics, and production of biofilm.Despite these efforts, solutions remain elusive.

Perhaps
the next generation of antimicrobials will include methods of targeting the
specific genes and regulatory mechanisms in bacteria that allow them to produce
biofilms.A combination of
biofilm-emulsifying drugs and good antibiotics will help us take giant steps
forward in our treatment of urologic infections.

Bacterial biofilms play an important role in the pathogenesis, persistence, and
eventual treatment of urinary tract infections (UTIs). Bacterial biofilms are
associated with catheter-associated UTIs, struvite calculogenesis, and chronic
prostatitis, as well as other common UTI scenarios. The bacterial biofilm theory
describes bacterial populations in natural and pathogenic ecological systems in
terms of a floating or "planktonic" population of bacteria interacting
with a more important matrix-enclosed "sessile" population of bacteria
associated with or adherent to a surface. This theory helps explain some of the difficulties encountered in the diagnosis and treatment of many
UTIs.

Introduction

The relatively new theory of bacterial biofilm helps clinicians understand and
perhaps explain some challenging areas in the pathogenesis, diagnosis, and
treatment of urinary tract infections (UTIs). The urinary tract is a hostile
environment for bacteria. Except for the distal urethra, it usually remains
sterile. Most UTIs are caused by ascending colonization and/or infection by
enteric bacteria of the perineum (in women, the vagina and introitus), the
periurethral area, the urethra, the bladder, and, occasionally, the kidney.
Infection results when the bacterial virulence factors overcome the numerous
host defenses.[1] The biofilm mode of growth allows bacteria to exist in the
urinary tract, resulting in many of the clinical infections.

Bacterial Biofilm

Direct microscopic observation has shown that many types of UTIs (eg,
catheter-associated infections, struvite urolithiasis, continuous ambulatory
peritoneal dialysis, and chronic prostatitis) are associated with biofilms
(adherent populations of bacteria).[2,3] Biofilm formation occurs when
microorganisms attach to a surface and, through growth and continuing
colonization, spread over that surface. Bacterial attachment is facilitated by
adhesins (structures on the bacterial cell surface, notably pili and
extracellular polymeric substances [EPS]), whose primary function is
adhesion.[4,5] As adherent cells grow, they form encapsulated microcolonies,
which are small clumps of morphologically identical cells (often 2-10 cells)
immediately
adjacent to each other. Growth of adjacent microcolonies toward each other will
lead to the development of a mature biofilm. This process and other aspects of
biofilm biology are presented in greater detail elsewhere.[6]

"Sessile" bacteria within biofilms are physiologically quite distinct
from unattached, "planktonic" bacteria. From a clinical perspective,
the most significant features of biofilms are the resistance of the component
microorganisms to antibiotics[7] and the immune response[8,9]; the creation of
chemically distinct microenvironments (within which microorganisms can form

calculi)[10]; and the potential for biofilm organisms to disseminate to other
organs such as the kidney.[11] The biofilm mode of growth enables bacteria to
persist in the urinary tract, but it is not unique to this system and occurs
naturally in other environments.[12]

From a clinical perspective, antibiotic resistance is the most problematic and
costly characteristic of biofilms. In vitro,[7,13] animal,[14] and clinical
data[15] have confirmed that bacterial growth within thick biofilms adherent to
urinary catheter material in a urine milieu confers a measure of antibiotic
resistance on the sessile bacteria cells within the biofilm. Planktonic, or
floating, cells in all these systems are completely eradicated at the antibiotic
levels predicted by laboratory MIC studies. However, more than 100 times the MIC
of antibiotics is required to eradicate cells within the bacterial biofilm.

Dispersion of the bacteria in the antibiotic-resistant biofilm further shows
that the individual bacteria remain susceptible to low levels of antibiotics
once they lose the protection of the biofilm itself. The increased resistance to
antibiotic therapy in such biofilms may be secondary to poor antibiotic
penetration into the biofilm matrix itself or decreased metabolic activity of
bacteria deep within the biofilm, especially during periods of environmental
(antimicrobial) threat.

Examination of biofilm structure (Fig. 1) provides a key to understanding
certain features of biofilms. At first glance, a biofilm appears to consist of a
copious, disordered mass of individual EPS-encased bacteria that adhere to a
surface. However, investigations of biofilms by scanning confocal laser
microscopy (SCLM) and other techniques[16] have shown their structure to be
quite complex, often containing mixed populations of bacteria. Current research
also suggests that cells within biofilms may actually communicate with each
other, using cell-density signaling molecules, such as acyl homoserine lactone,
for the purpose of coordinating metabolic activities and responses to stressors
such as antibiotics.[17] Bacterial cells at the periphery of a biofilm, or those
immediately adjacent to the water channels that separate them, are
faster-growing than those within a microcolony because of greater access to
nutrients. However, these faster-growing cells are more susceptible to attack
by components of the immune system (such as lymphocytes) or by antibiotics than
are the slower-growing cells buried within the microcolonies. Metabolic activity
within a biofilm may also create a chemical microenvironment (eg, one conducive
to struvite formation) that may be considerably different from that of the
larger environment surrounding it.

Catheter-Associated Infections

Bacterial biofilm has been associated with biomaterial-related sepsis such as
that transmitted by urinary Foley catheters. Improved understanding of the
pathogenesis of catheter-associated UTIs and the role of bacterial biofilms
helps explain why catheterization is the most common cause of nosocomial
infection in medical practice despite major technologic changes in catheter
material, design, and collecting systems. In vitro,[18] animal,[19] and clinical
studies[20] have shown that the pathogenesis of these infections involves an
ascending, or creeping, bacterial biofilm. Observations from these experimental
models have shown that bacteria form thick, coherent biofilms adherent to
contaminated drainage spouts extending proximally into the drainage bag and
subsequently into the catheter (Fig. 2). If a sterile, closed system is strictly
maintained, the extraluminal route from the urethral meatus becomes the
predominant route of catheter-associated bacteriuria.
In the absence of antibiotics, it appeared that the ascending bacterial biofilm
moved by 2 mechanisms: (1) rapidly dividing bacteria cells spreading along the
catheter surface within the glycocalyx material of the biofilm and (2)
planktonic or floating bacteria cells within the urine column leapfrogging just
ahead of the adherent biofilm, perhaps assisted by the turbulence caused when
the urine flow meets the biofilm front (saltatory movement).

The
bacterial populations demonstrate a heterogeneity that is not evident from the
culture results. Only a small proportion of the microorganisms including fungi,
which are identified morphologically by scanning or transmission electron
microscopy, are recovered by routine culture method. The nature of the bacterial
biofilm adherent to the catheter can be appreciated by aspiration cultures of
the planktonic bacteria being released from the biofilm. Usually, however, at
this point in the process the bacteria are colonizing only the catheter surface
and have not yet caused cystitis. As the adherent bacterial aggregate becomes
larger, the now macroscopic bacterial biofilm can create flow problems by
partially blocking catheter inlets and reducing the tubular diameter of the
catheter lumen.

Planktonic bacteria are continually shed from the colonized catheter into the
residual urine that is always present around the tip and balloon of the
catheter. The final step from asymptomatic bacteriuria to symptomatic,
catheter-associated cystitis involves actual adherence of these bacteria to the
bladder surface. The indwelling Foley catheter appears to disrupt the bladder
mucus or glycosaminoglycan layer by causing mechanical or chemical irritation
and even erosion of the bladder mucosa, exposing surfaces that allow bacterial
adherence. The synergistic activity between antibiotics and host defenses in
these infections appears to clear the mucosal surfaces of adherent bacterial
microcolonies much more easily than they are able to clear the very resistant
bacterial biofilms adherent to the inert biomaterial of the catheter.[14]

Although the biofilm theory has helped explain difficult, Foley
catheter-associated infection, currently the most effective means of reducing
the incidence of these infections is to avoid indwelling, chronic
catheterization, if at all possible, or at least to reduce the length of time
the catheter remains in the bladder. In the future, new biomaterial research may
allow production of materials that reduce bacterial biofilm attachment and
enhance mucosal biocompatibility. New antibiotics are being developed that may
be able to penetrate the bacterial biofilm.

Infection Stones of the Urinary Tract

Proteus mirabilis and other urease-producing bacteria are major causes of
UTIs.[21,22] The major risk in these UTIs is the development of urinary calculi,
which typically contain struvite (NH4MgPO4.6H20) and carbonate-apatite
(Ca10(PO4,CO3)6(OH,CO3)2) as the predominant mineral components. Infection
stones account for only 5% to 20% of all urinary calculi; however, they
represent a much more serious threat to the organs of the urinary tract than do
conventional metabolic stones because of their rapid growth and high rate of
recurrence. By blocking catheters and stents, infection stones cause both direct
renal damage as well as secondary effects.[23,24] Griffith and colleagues[25]
illustrated the fundamental role of urease in the pathogenesis of this
infection. Urea hydrolysis by bacterial urease activity elevates urine pH and
leads to Mg and Ca precipitation in the form of struvite and other minerals.
Ultrastructural examination of infection stones reveals the
growth of microorganisms throughout struvite calculi (Fig. 3).[26]

It has been proposed that the bacterial biofilm mode of growth and its organic
matrix (EPS) are largely responsible for initiating matrix deposition and
crystal binding (within the urinary tract) and may be crucial in the process of
crystal nucleation through creating an alkaline, metal (Ca and Mg)-saturated
microenvironment.[27,28] Mature struvite stones have been likened to
"fossilized biofilms"[29] within which the causative microorganisms
are shielded from the effects of antimicrobial agents.[30] Of equal or greater
importance, any dislodged or residual calculus fragments would contain viable
organisms and could therefore act as seeds for the rapid development of new
calculi. These 2 features explain the high recurrence of these calculi (about
50%) in spite of conventional surgical techniques.[21]

The knowledge of how struvite calculi are formed may encourage use of specific
treatment measures, such as effective urease inhibition, and may also serve to
emphasize the absolute necessity of removing all stone fragments and eradicating
all organisms associated with the UTI. Otherwise the stones can and will recur.

Chronic Bacterial Prostatitis

Although the diagnostic and classification systems in chronic prostatitis have
been standardized, difficulties in differentiating chronic nonbacterial from
bacterial prostatic inflammation are encountered in clinical practice.
Additionally, the results of antibiotic treatment guided by culture and
antibiotic-sensitivity data are dismal.[31,32] Current research on the
association of bacterial biofilms associated with chronic prostatitis is
unlocking some of the mystery surrounding this entity and will allow us to
further rationalize diagnostic and therapeutic regimens.[33] It appears that
bacteria in chronic bacterial prostatitis enter the prostate gland from the
urethra (as ascending infection), perhaps assisted by turbulent urethral flow
patterns and/or intraprostatic ductal reflux. Clinical studies involving a
difficult group of patients with bacterial prostatitis who remain refractory to
therapy have led to a further understanding of what is actually happening in the
prostate gland during acute and chronic bacterial prostatitis.[33]

Once the bacteria enter the ducts and ascini of the prostate gland, they rapidly
multiply, inducing a host response with infiltration of acute inflammatory cells
into the ducts. In acute bacterial prostatitis, the entire prostate gland, or at
least the major part of it, is involved in the inflammatory process. The ducts
become engorged with infiltrate composed of dead and live bacteria as well as
living and dying acute inflammatory cells, desquamated epithelial cells, and
cellular debris. At this stage of the infection, because the majority of cells
are planktonic, it is relatively easy to eradicate all the offending organisms
with appropriate antibiotic therapy for complete resolution of the inflammatory
process.

If bacteria persist from acute or, more likely, clinically subacute
inflammation, they can form small, sporadic bacterial microcolonies or biofilms
within the ductal system adherent to the epithelium (Fig. 4). These bacteria
also produce an exopolysaccharide slime, or glycocalyx, that envelops these
adherent microcolonies, and it appears that the microorganisms subsequently
become very quiescent, undergoing a sort of "hibernation" when the
environment becomes threatening. Surrounding these focal sites of bacterial
persistence are areas of lymphocytic invasion with variable infiltration of
plasma cells and macrophages. It appears that the persistence of bacteria in the
prostate gland in these focal biofilms leads to persistent immunologic
stimulation and subsequent chronic inflammation.

The traditional diagnostic routine -- although difficult, time-consuming, and
expensive -- is the absolute key to diagnosis. It employs quantitative bacterial
cultures of various urine segments and expressed prostatic secretion from the
lower urinary tract.[31] However, urologists have abandoned this theoretically
effective diagnostic culture technique because of a number of
identifiable shortcomings.[32] The primary problem is that previous antibiotic
therapy (prescribed prior to obtaining the proper specimens) appears to mask
subsequent attempts at bacterial localization. Antibiotic therapy eradicates the
planktonic bacteria within the ducts, and the very adherent bacteria in the
biofilms do not appear to shed planktonic bacteria easily. This finding has been
subsequently confirmed by biopsies of the prostate glands of prostatis patients
with negative expressed prostatic secretion cultures when the organism is
nonetheless detected within the prostate gland tissue culture.[33,34]
Bacterial biofilms also appear to influence treatment results. The
pharmacokinetics in noninflamed prostate glands have identified a number of
antibiotics, including trimethoprim and the quinolones, as agents that can
achieve reasonable levels within the prostatic fluid. Detailed pharmacokinetic
studies of prostatitis in a rat model failed to show any significant difference
in antibiotic levels in the inflamed prostatic duct.[35] It is much more likely
that the adherent glycocalyx-encased, bacterial-biofilm mode of growth is
conveying a relative resistance to the associated bacteria similar to that seen
in catheter- and struvite-associated UTIs.

The knowledge that small adherent bacterial biofilms can exist deep within the
prostate gland in chronic bacterial prostatitis should improve diagnostic and
treatment regimens. It appears that an immunologic diagnosis -- based on the
premise that although we cannot grow planktonic organisms, the sessile organisms
continue to create an immune response that can be measured -- may improve
differentiation of nonbacterial from bacterial prostatitis.[36,37]

Treatment regimens that deliver much higher antibiotic concentrations to the
biofilm itself in the prostatic duct, theoretically improving treatment success
rate (J.C. Nickel, unpublished date from ongoing investigation), are being
developed. Finally, repetitive prostatic massage -- the historic treatment of
prostatitis -- is making a comeback with clinicians.[38] Regular prostatic
massage drains the obstructed ducts and perhaps converts more resistant sessile
biofilm bacteria into sensitive planktonic forms that then become susceptible to
treatment with newer, more potent antibiotics.

Biofilms Associated with Other UTIs

Urethritis/Cystitis

It is generally accepted that the route of infection and the development of
cystitis begins with intestinal bacteria colonizing the introitus and the
periurethral area with subsequent invasion of the bladder. An indigenous
population of gram-positive bacteria, such as Lactobacillus species, exists
within the introitus and vagina. They form a type of thin, living biofilm or
bacterial barrier on the mucosa, which competitively inhibits the growth of
pathogenic gram-negative bacteria.[1] However, if the protected bacterial
population is defective, the invading pathogens may displace the favorable
population and adhere to the introitus and periurethral area.

From colonization (as a thin biofilm on the periurethral and distal urethra) the
bacteria instigate an ongoing battle against the intrinsic urinary tract
defenses. When bacteria enter the bladder, individual floaters or planktonic
bacteria are susceptible to the hostile environment of the urine, the mechanical
forces of urine evacuation, and the bladder surface itself. If circumstances
favor bacterial virulence over host defenses, uropathogens adhere to the mucosal
surface in small, sporadic aggregates or microcolonies. These eventually may
coalesce in patchy areas of bacterial biofilm adhering to superficial epithelial
cells. At this point in the process, the resulting inflammation produces
cystitis. Small doses of antibiotics prevent bacterial adherence and subsequent
early biofilm formation and may be considered a prophylactic strategy.

Pyelonephritis

When vesicoureteral reflux occurs, planktonic bacteria in the bladder secondary
to asymptomatic bacteriuria or bacterial aggregates in cystitis can be flushed
into the kidney. Alternatively, bacteria may ascend as a biofilm along the inner
wall of a ureter paralyzed by the bacterial inflammatory process. Again, if
bacterial virulence outweighs the host defenses of the kidney or if further
defects such as renal scarring are present, the planktonic bacteria may adhere
to the urothelium and papilla of the renal collecting system. Employing the P
mirabilis model,[11] we have demonstrated that these bacteria may adhere in thin
biofilms to the uroepithelium prior to invasion of the renal tissue and
subsequent pyelonephritis.

Other Prosthesis-Related UTIs

An indwelling ureteric stent can also develop a bacterial biofilm that can act
as a continuing nidus for infection or that can build up to cause subsequent
obstruction.[39] Penile prostheses used to treat impotence and external
sphincter prostheses used to treat incontinence can also become
secondarilyinfected. These infections are associated with bacterial biofilm
attached to
the biomaterial surface.[40] Similarly, infectious complications associated with
devices used in nephrology (eg, peritoneal dialysis catheters, femoral and
subclavian central venous catheters) are associated with bacterial biofilms.[41]
These infections are not only resistant to antimicrobial therapy but can also
cause life-threatening blockage of vital lines and tubes.

Summary

Bacterial biofilms are implicated in the more serious and difficult UTIs
encountered by clinicians in daily practice. Biofilm theory would attest to the
futility of antibiotic therapy in these infections associated with inert
prostheses (such as stents, catheters, and prostheses), and clinical experience
has shown that the infections can eventually be successfully eradicated only by
removal of the prostheses. Biofilms associated with obstructed prostate ducts in
chronic prostatitis are problematic. The tenuous bacterial biofilms associated
with cystitis and, perhaps, early pyelonephritis appear to be more easily
eradicated by antibiotics compared with surface biofilms such as those
associated with catheters because of the synergistic activity of antibiotics and
host defenses against these bacterial biofilms. Understanding the bacterial
biofilm concept and its association with host response and inert prostheses
within the urinary tract is important with regard to improving diagnostic and
therapeutic strategies in the management of these difficult UTIs.

If a
document is too wordy, trying searching the document ("control f" on
your keyboard) for prostatitis, then prostate, then biofilm - 3 separate
searches. The search will take you to the word you are searching for.

An
unusual organism called Nanobacterium sanguineum has been identified in
association with prostatic and other calcifications, although some
controversy remains surrounding the characteristics and pathogenicity of this
ultra-small and difficult to detect bacterium. It is unusual in many
respects including its small size (about 1/1000 of the usual bacterial size) and
its uniform association with layers of calcification, called a biofilm, with
which it encapsulates and protects itself. It is believed by some to be associated with virtually all forms
of calcification in the human body including prostatic calculi (stones), kidney
stones, calcification of the ocular lens (cataracts) and arterial calcification
(coronary and carotid artery disease). It has recently been shown to give a
false positive test for Chlamydia. Very few labs are equipped to detect this
organism and most physicians have still not heard of
it.

There
is a commercial product being tested for treatment of coronary disease which
uses a nightly rectal suppository of EDTA (this chelates calcium and unroofs the
nanobacter making it vulnerable to the antibiotic) combined with oral enzymes
and certain vitamins that augment the EDTA, and a bedtime dosage of
tetracycline (doxycycline won't work) which kills the nanobacter once it is
unroofed. The NanobacTx regimen is
proprietary and only available through the group that developed it. You need to
go to their website as I suggested and contact them to find out if any doctor in
your area is able to write you the prescription for their program.

Does it work for
prostatitis? Anecdotally, the answer is yes. I tried it for myself
since I have well documented prostatitis. I had severe, longstanding and
intractable pain with ejaculation, and the urologists were unable to explain it
or treat it effectively. It has made a huge difference for me.

The 2
researchers from Finland who discovered the Nanobacterium sanguineum by serendipity and
discovered its association with calcification were nominated for the Nobel
prize in 2000 and are actively engaged in expanding the knowledge base.

Nanobacteria are
novel apatite mineral-forming agents found in human and animal blood and
tissues, and arouse an antibody response. Earlier studies have shown that
antibodies that react with nanobacteria also cross-react with bacteria from the
related Bartonella group. To study possible cross-reactions further we tested
serum samples from 400 Swedish healthy blood donors for nanobacterium and
Bartonella antibodies.

Antibodies to
nanobaceria were assayed using an ELISA test supplied by Oy Nanobac Ltd (Kuopio,
Finland). Bartonella IFA was performed using antigen preparations of
Vero-cells co-cultivated with B henselae , B. henselae (”Marseille-strain”),
B. elizabethae, B. quintana and B. grahamii. Four hundred serum samples were
obtained from local blood banks at regional hospitals in Lund, Jönköping,
Uppsala and Boden. The samples were collected during 1999 from healthy
volunteers. Age and sex, as well as information on recent animal contacts were
recorded in 393 cases. Of the 400 samples, 56 were positive (56/400 = 14%), using a cutoff value of 2 times
the average of 4 negative controls. Seventeen of the samples were highly
positive (17/400 = 4.25%) (> 3 times the average of negative controls).

There was no
statistically significant overlap between Bartonella- and nanobacterium-positive
serum samples. Furthermore, there was no significant overrepresentation of
gender, any age group or geographical locality in the donors who had
nanobacterial antibodies. However, counting only the strongly positive serum
samples, there was a significant overrepresentation in those donors that had
recent contact with certain animals. The strongest association was seen in those
with horse contact (p=0.007), but also in sheep (p=0.012) and cow contacts
(p=0.026).

In conclusion, we
found no cross-reactions of serum samples positive to nanobacteria with five
different Bartonella antigens. Our results thus indicate separate risk factors
for exposure to Bartonella and nanobacteria. At this point they are largely
unknown, but it is interesting to note that the most strongly seropositive cases
to nanobacteria were associated with animal contacts.